A microscope with evanescent illumination of a sample is known from US 2002/0097489 A1. The microscope comprises a white light source, the light of which is coupled for the purpose of evanescent illumination via a slit aperture through the microscope objective onto a sample holder, which holds a sample. The illumination light propagates itself in the sample holder by means of total internal reflection, whereby the illumination of the sample occurs only in the region of the evanescent field that protrudes from the sample holder. Microscopes of this type are known as “total internal reflection fluorescent microscopes” (TIRFM).
The z-resolution of TIRF microscopes is extraordinarily good because the evanescent field protrudes only about 100 nm into the sample.
A high-aperture objective specifically for TIRF application is known from DE 101 08 796 A1. The objective comprises a first lens with positive refractive power and a second lens with negative refractive power, whereby the focal distance ratio between the two lenses is in the −0.4 and −0.1 range, and the total refractive power is greater than zero. The objective further comprises two positive lenses, the diameter ratio to focal length of which is greater than 0.3 and less than 0.6. The objective further comprises a negative lens and a collecting lens, whereby the negative lens faces the front group, and the focal distance ratio of the negative lens to the collector lens is between −0.5 and −2.
An incident illumination device for TIRF microscopy is known from DE 102 17 098 A1. The incident illumination device comprises an illumination source that emits a polarized illumination beam when in operation, which propagates at an angle to the optical axis and a deflector that deflects the illumination light beam and couples it parallel to the optical axis in the objective. Provision is made in this incident illumination device for the illumination light beam emitted by the illumination source to exhibit a phase difference in the s- and p-polarization directions, and for the deflection arrangement to reflect the illumination light beam x times, whereby x=(n×180°−d)/60°.
A microscope for total internal reflection microscopy (TIRM) is known from DE 101 43 481 A1. The microscope exhibits a microscope housing and an objective. The illumination light emitted by an illumination device can be coupled via an adapter that can be inserted into the microscope housing.
A microscope with an optical illumination system that enables simple switching between evanescent illumination and reflective illumination is known from US 2004/0001253 A1. The illumination system comprises a laser light source, the light of which is coupled in an optical fiber. Furthermore, an outcoupling optic is provided that focuses the light that exits from the fiber onto a rear focal point of the microscope objective. The optical fiber is movable along a plane that is perpendicular to the optical axis of the microscope objective.
A device for coupling light in a microscope is known from DE 102 29 935 A1. Here, a laser light is directed onto a sample in the illuminated field diaphragm plane by a laser light fiber coupling, which is implemented as a slide. The invention is particularly suitable for the TIRF method.
In scanning microscopy, a sample is illuminated with a light beam to observe the detection light emitted by the sample as reflection or fluorescent light. The focus of an illumination light beam is moved on an object plane with the help of a movable beam deflector, generally by tipping two mirrors, whereby the axes of deflection are usually positioned perpendicular to each other, so that one mirror deflects in the x-direction and the other in the y-direction. The mirrors are tipped with the help, for example, of galvanometric positioners. The power of the light coming from the object is measured dependent on the position of the scanning beam. Generally, the positioners are provided with sensors to determine the actual position of the mirrors. In confocal scanning microscopy in particular, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optic with which the light from the source is focused on a pinhole aperture—the so-called excitation aperture—, a beam splitter, a beam deflector to control the beam, a microscope optic, a detection aperture, and detectors to detect the detection light or fluorescent light. The illumination light is coupled via a beam splitter. The fluorescent light or reflected light emitted by the object returns to the beam splitter via the beam deflector, passes through it, and is subsequently focused onto the detection aperture, behind which are located the detectors. This arrangement of detectors is called a descan arrangement. Detection light that does not originate directly from the focal region takes another light path and does not pass through the detection aperture so that pixel information is obtained, which is converted into a three-dimensional image by sequential scanning of the object with the focus of the illumination light beam. A 3-dimensional image is generally achieved by layered imaging.
Arrangements that increase the resolving power of a confocal scanning microscope are, among other things, given by the intensity distribution and the spatial expansion/extent/extension of the focus of the excitation light beam. An arrangement for increasing the resolving power for fluorescence applications is known from PCT/DE/95/00124. Here, the lateral peripheral areas of the focus volume of the excitation light beam are illuminated with a light beam of a different wavelength, the so-called stimulation light beam, which is emitted from a second laser, in order to return the sample areas stimulated there by the light of the first laser to their normal state. Only the light emitted spontaneously from the areas not illuminated by the second laser is detected so that an overall improvement in resolution is achieved. This method is known as STED (stimulated emission depletion).
For example, a variant of the STED method is known from US 2002/0167724 A1 and from US 6,667,830 B1, in which the sample areas that are excited by the light from the first laser are next excited by the light of the second laser—into a third state. In this variant, known as “up-conversion,” increases in resolution are achieved equivalent to those achieved by the variant with directly stimulated de-excitation to the normal state.
Coherent anti-Stokes Raman (CARS) microscopy is an art that is gaining in importance. One great advantage is that the samples do not need to be marked with dyes. Furthermore, living cells may be tested.
In comparison with conventional Raman microscopy and known confocal Raman microscopy, CARS microscopy makes it possible to achieve higher detection light yield, better suppression of interference, and greater ability to separate detection light from illumination light. A detection pinhole and a high-resolution spectrometer are needed in conventional confocal Raman spectroscopy in order to achieve good axial resolution. CARS, by contrast, is a nonlinear optical process (four-wave mixing process). In a manner similar to multi-photon microscopy, in which two or more photons are absorbed simultaneously, no detection pinhole is needed because the probability of in-phase simultaneous convergence of several photons in the focus is at its greatest because of the high photon density. The same axial resolution as with multi-photon microscopy is achieved without a detection pinhole. In CARS spectroscopy, 2 lasers that emit light at different wavelengths are usually used (νP and νS, Pump and Stokes laser), whereby νS should be tunable in order to produce (νCARS=2νP-νS, ICARS˜(IP)2.IS) a CARS spectrum νCARS. If the difference frequency νP-νS agrees with the difference frequency between two molecular vibration states |1 and |0 in the sample, the CARS signal is actually strengthened. The pump light beam and the Stokes light beam are coaxially combined in microscopic applications and are together focused on the same sample volume. The direction in which the anti-Stokes beam is emitted results from the phase-matching condition for the four-wave mixing process.
A device is known from U.S. patent application Ser. No. 4,405,237, “Coherent anti-Stokes Raman device,” in which two pulsed laser beams, which are produced by two lasers, and which exhibit different wavelengths in the visible region or in the UV region of the spectrum, are used to illuminate a sample simultaneously. With appropriate wavelength selection, the sample may be excited such that it emits the characteristic coherent anti-Stokes Raman beam.
A method for exciting an evanescently illuminated sample using a two-photon process is known from James W. M. Chon, Min Gu, “Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: Image formation,” Appl. Opt. 43, 1063-1071, 2004, and from Florian Schapper, José T. Gonçalves, Martin Oheim, “Fluorescence imaging with two-photon evanescent wave excitation,” Eur. Biophys. J. 32, 635-643, 2003.
The previous art for evanescent sample illumination merely enables the testing of sample layers that directly adjoin the cover glass or the sample holder.